Importance of antibody and complement for oxidative burst and killing of invasive nontyphoidal Salmonella by blood cells in Africans

Abstract

Bacteremia caused by nontyphoidal strains of Salmonella is endemic among African children. Case-fatality rates are high and antibiotic resistance increasing, but no vaccine is currently available. T cells are important for clearance of Salmonella infection within macrophages, but in Africa, invasive Salmonella disease usually manifests in the blood and affects children between 4 months and 2 y of age, when anti-Salmonella antibody is absent. We have previously found a role for complement-fixing bactericidal antibody in protecting these children. Here we show that opsonic activity of antibody and complement is required for oxidative burst and killing of Salmonella by blood cells in Africans. Induction of neutrophil oxidative burst correlated with anti-Salmonella IgG and IgM titers and C3 deposition on bacteria and was significantly lower in African children younger than 2 y compared with older children. Preopsonizing Salmonella with immune serum overcame this deficit, indicating a requirement for antibody and/or complement. Using different opsonization procedures, both antibody and complement were found to be necessary for optimal oxidative burst, phagocytosis and killing of nontyphoidal Salmonella by peripheral blood cells in Africans. Although most strains of African nontyphoidal Salmonella can be killed with antibody and complement alone, phagocytes in the presence of specific antibody and complement can kill strains resistant to killing by immune serum. These findings increase the likelihood that an antibody-inducing vaccine will protect against invasive nontyphoidal Salmonella disease in African children.

Fig. 1.

Oxidative burst in neutrophils in whole blood from Malawian children following stimulation with Salmonellae. Neutrophil oxidative burst to unopsonized (A–D) and preopsonized (E–H) S. Typhimurium D23580 compared with (A and E) age of the child, (B and F) anti–S. Typhimurium D23580 IgG titer, (C and G) anti-D23580 IgM titer, and (D and H) C3 deposition on D23580. Each point corresponds to blood from one child (N = 64).

Fig. 2.

Effect of different opsonization procedures on oxidative burst activity in African adult washed peripheral blood phagocytic cells following stimulation with Salmonellae. Oxidative burst in neutrophils (A and B) and monocytes (C and D) following stimulation with immune serum–sensitive S. Typhimurium D23580 (A and C) and immune serum–resistant S. Typhimurium D26104 (B and D). NC, negative control, no stimulant; unop, unopsonized Salmonellae; ab-def, Salmonellae opsonized with anti-Salmonella antibody-deficient serum; immune, opsonized with immune serum; C6-def, opsonized with C6-deficient serum; HI, opsonized with heat-inactivated serum; IgG, opsonized with IgG purified from immune serum; Monos, monocytes; Neuts, neutrophils. Points in each group of four are from separate experiments. *P ≤ 0.0001; ^†P < 0.002.

Fig. 3.

Effect of different opsonization procedures on phagocytosis of Salmonellae by African adult washed peripheral blood cells. Percentage of washed neutrophils (A and B) and monocytes (C and D) phagocytosing immune serum–sensitive S. Typhimurium D23580 (A and C) and immune serum–resistant S. Typhimurium D26104 (B and D). Abbreviations as for Fig. 2. Points in each group of four are from separate experiments. *P < 0.0001; ^†P < 0.01.

Fig. 4.

Effect of different opsonization procedures on killing of Salmonellae by African adult washed peripheral blood cells. In vitro killing of (A) serum-sensitive S. Typhimurium D23580 and (B) serum-resistant S. Typhimurium D26104 at 45, 90, and 180 min. Negative values correspond with a decrease in viable Salmonellae compared with the initial concentration of 10⁵ cfu/mL Abbreviations as for Fig. 2: Unop (filled circle, solid line); ab-def (filled triangle, solid line); immune (filled square, solid line); C6-def (empty square, dashed line); HI (empty triangle, dashed line); IgG (empty circle, dashed line). Data are mean ± SD of three experiments.